The capsular polysaccharide-protein conjugate vaccine is effective in prevention of invasive disease by Streptococcus pneumoniae (pneumococci) of the serotypes included but is costly to make and administer, minimally effective against otitis media, and subject to serotype replacement (in which non-included serotypes become more prevalent). Therefore, novel approaches to immunization are needed. We found unexpectedly that mice immunized intranasally with the pneumococcal cell wall polysaccharide (CWPS, an antigen commonto all serotypes) develop long-lasting resistance to nasopharyngeal colonization and middle ear infection with pneumococci of different serotypes. Strikingly, protection by the vaccine(i.e. CWPS + mucosal adjuvant) is independent of antibody and dependent on the presence of CD4+ T cells. This polysaccharide-induced, cell-mediated mucosal immunity against colonization by an "extracellular" encapsulated bacterium has not, to our knowledge, been previously demonstrated and therefore represents a novel approachto vaccination. We hypothesize that the zwitterionic property of CWPS is critical in eliciting this T-cell-dependent response. Our first goal is to determine the structural basis of protection by CWPS by further purification, polymer synthesis, chemical modifications to alter the zwitterionic motif, auto-coupling, or coupling to a protein carrier. Thus either the zwitterion hypothesis will be confirmed and/or the minimal protective structure will be defined. Secondly, we will examine in more detail the mechanisms whereby CD4+ T cells confer protection against pneumococcal colonization. Adoptive transfer experiments will characterize the nature of the protective T cell responses. Further approaches will include polarization of T cell responses by use of knockout mice or administration of cytokines, neutrophil depletion experiments, and histopathology with confocal microscopy. In a third aim, we will evaluate the role of innate immune responses in modulating acquired immunity to colonization, by use of Toll-like receptor (TLR) knockout mice and co-administration of TLR ligands as adjuvants at the time of immunization. Our studies will increase basic understanding of immunity to pneumococcal colonization and could lead to a simple, defined, and possibly synthetic vaccine that would complement or replace the multivalent capsular conjugates in vaccination against this, highly prevalent pathogen of children.